Regulation of membrane phospholipid syntheesis in Escherichia coli during temperature up-shift.

The synthesis of membrane phospholipids and that of stable ribonucleic acid were inhibited during temperature up-shift of both rel+ and relA strains of Escherichia coli. The kinetics of the inhibition of the synthesis of both molecules were correlated with the kinetics of guanosine 5'-diphosphate-3'-diphosphate synthesis. Metabolic down-shift experiments gave similar results.     

Fermentative Accumulation of Guanosine Polyphosphate by Brevibacterium ammoniagenes

Guanosine-3'-diphosphate-5'-monophosphate (3.35 mg/ml), guanosine-3'-diphosphate-5'-diphosphate (MSI) (5.21 mg/ml), and guanosine-3'-diphosphate-5'-triphosphate (MSII) (0.82 mg/ml), in addition to guanosine 5'-monophosphate, guanosine 5'-diphosphate, and guanosine 5'-triphosphate, were accumulated by microbial conversion of 5'-xanthylic acid with a mutant of Brevibacterium ammoniagenes.     

Nitrogenase synthesis in Klebsiella pneumoniae: enhanced nif expression without accumulation of guanosine 5'-diphosphate 3'-diphosphate

Derepression of nitrogen fixation (nif) genes in Klebsiella pneumoniae following transfer from NH+4-sufficiency to N-free medium was preceded by rapid expansion of the guanosine 5'-diphosphate 3'-diphosphate (ppGpp) pool. When derepressed in N-free medium supplemented with glutamine (600 micrograms ml-1), expression from the nifH and nifL promoters, determined as beta-galactosidase activity in nif::lac merodiploid strains, was stimulated 7-fold and nitrogenase activity 26-fold; ppGpp did not accumulate, remaining at the levels found in NH+4-repressed populations. The relaxed mutant K. pneumoniae relA40, which accumulates only very low levels of ppGpp, showed partial derepression of nitrogenase activity in the presence of glutamine, thus ppGpp is unlikely to be an effector of nif expression. ATP and GTP levels were elevated under conditions where nif expression was enhanced, consistent with previous data suggesting that maintenance of ATP levels is a prerequisite for the expression of nif genes in K. pneumoniae.     

Influence of amino acid starvation on guanosine 5''-diphosphate 3''-diphosphate basal-level synthesis in Escherichia coli.

We observed that the synthesis of basal-level guanosine 5'-diphosphate 3'-diphosphate (ppGpp) in both relA mutants and relA+ relC strains of Escherichia coli decreased in response to amino acid limitation and that this was accompanied by an increase in ribonucleic acid (RNA) synthesis. Addition of the required amino acid to starved cultures of relaxed bacteria resulted in the resumption of ppGpp synthesis and a concomitant decrease in RNA production. Our results indicate that relA mutants retain a stringent factor-independent ribosomal mechanism for basal-level ppGpp synthesis. They also suggest that in relA+ bacteria, stringent factor-mediated ppGpp synthesis and the production of basal-level ppGpp are mutually exclusive. These findings substantiate the hypothesis that there are two functionally discrete mechanisms for ppGpp synthesis in E. coli. Through these studies we have also obtained new evidence which indicates that ppGpp serves as a modulator of RNA synthesis during balanced growth as well as under conditions of nutritional downshift and starvation.     

A new nucleotide involved in the stringent response in Escherichia coli. Guanosine 5'-diphosphate-3'-monophosphate.

A novel nucleotide has been detected in Escherichia coli subjected to the stringent response. However, this nucleotide does not accumulate in relA+ cells subjected to heat shock, in which guanosine 5'-diphosphate-3'-diphosphate does accumulate but stable RNA synthesis is not restricted. The intracellular level of this new nucleotide thus correlates well with control of stable RNA synthesis. Chemical and enzymatic analysis shows that the new nucleotide is guanosine 5'-diphosphate-3'-monophosphate. It is suggested that this nucleotide may play a role in stringent control of stable RNA synthesis.     

Protein synthesis results in guanosine-5'-diphosphate-3'-diphosphate synthesis in Escherichia coli minicells.

Minicells of Escherichia coli DS410 synthesized guanosine-5'-diphosphate-3'-diphosphate and guanosine-5'-triphosphate-3'-diphosphate when synthesizing proteins in response to phage T7 infection. The guanosine-5'-diphosphate-3'-diphosphate synthesis was found to be relA+ dependent and inhibited by chloramphenicol.     

Near-UV stress in Salmonella typhimurium: 4-thiouridine in tRNA, ppGpp, and ApppGpp as components of an adaptive response.

We have examined the role of 4-thiouridine in the responses of Salmonella typhimurium to near-UV irradiation. Mutants lacking 4-thiouridine (nuv) and mutants defective in the synthesis of ppGpp (guanosine 5'-diphosphate-3'-diphosphate) (relA) were found to be sensitive to killing by near-UV. Near-UV induced the synthesis of a set of proteins that were not induced in the nuv mutant. Some of these proteins were identified as oxidative defense proteins, and others were identified as ppGpp-inducible proteins. Over 100-fold increases in ApppGpp (adenosine 5', 5"'-triphosphoguanosine-3"'-diphosphate, the adenylylated form of ppGpp) were observed in wild-type cells after near-UV irradiation but not in the 4-thiouridine-deficient mutant. These data support a model in which ppGpp and ApppGpp, a dinucleotide proposed to be synthesized by tRNA-aminoacyl synthetases as a response to the cross-linking of 4-thiouridine in tRNA by near-UV, induce the synthesis of proteins necessary for resistance to near-UV irradiation.     

Mutation spoT of Escherichia coli increases expression of the histidine operon deleted for the attenuator.

F'-episomes carrying the Salmonella typhimurium wild-type or attenuator-deleted histidine (his) operons were introduced into Escherichia coli strains containing relA or spoT single and double mutations known to affect guanosine 3'-diphosphate 5'-diphosphate (ppGpp) and guanosine 3'-triphosphate 5'-diphosphate (pppGpp) levels. Expression of the his operon and expression of the gene for 6-phosphogluconate dehydrogenase (gnd) were measured during balanced growth in amino acid-rich and minimal media. The data were consistent with the interpretation that ppGpp is a positive effector of his operon expression, whereas pppGpp is not an essential effector. The conclusion that his operon expression is maximally stimulated at a lower than maximum intracellular ppGpp concentration was further confirmed. Neither ppGpp nor pppGpp appeared to influence gnd gene expression. The metabolic regulation of the E. coli his operon was found to be similar to the ppGpp-meidated metabolic regulation of the S. typhimurium his operon.     

Control of the activity of the soluble lytic transglycosylase by the stringent response in Escherichia coli

The soluble lytic transglycosylase (Slt) of Escherichia coli is known to be a powerful murein hydrolase in vitro. It is shown here to act as an autolysin in vivo as well. Rapid autolysis of Slt overproducing cells was induced by protein biosynthesis inhibitors, which also block the fomration of guanosine-5'-diphosphate-3'-diphosphate (ppGpp). When amino acid starvation was used to inhibit protein synthesis, autolysis was suppressed in relA+ but not in relA- cells. These findings indicate that the stringent control modulates the enzymatic activity of the soluble lytic transglycosylase in vivo.     

Stringent control of peptidoglycan biosynthesis in Escherichia coli K-12.

[3H]Diaminopimelic acid (Dap) was incorporated exclusively into peptidoglycan by Escherichia coli strains auxotrophic for both lysine and Dap. The rate of [3H]Dap incorporation by stringent (rel+) strains was significantly decreased when cells were deprived of required amino acids. The addition of chloramphenicol to amino acid-starved rel+ cultured stimulated both peptidoglycan and ribonucleic acid synthesis. In contrast, a relaxed (relA) derivative incorporated [3H]Dap at comparable rates in the presence or absence of required amino acids. Physiologically significant concentrations of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) inhibited the in vitro synthesis of both carrier lipid-linked intermediate and peptidoglycan catalyzed by a particulate enzyme system. The degree of inhibition was dependent on the concentration of ppGpp in the reaction mixture. Thus, the results of in vivo and in vitro studies indicate that peptidoglycan synthesis is stringently controlled in E. coli.     

Mutations in the spoT gene of Salmonella typhimurium: effects on his operon expression.

The spoT gene of Salmonella typhimurium has been identified. Mutations in spoT map between gltC and pyrE at 79 min. The spoT1 mutant has elevated levels of guanosine 5'-diphosphate-3'-diphosphate (ppGpp) during steady-state growth and exhibits a slower than normal decay of ppGpp after reversal of amino acid starvation. The spoT1 mutation elevates his operon expression but is distinct from known his regulatory mutations. Elevated his operon expression in spoT mutants causes resistance to the histidine analogs, 1,2,4-triazole-3-alanine and 3-amino-1,2,4-triazole. These properties of spoT mutants allowed us to identify and characterize additional spoT mutants. Approximately 40% of these mutants are temperature sensitive for growth on minimal medium, suggesting that the spoT function is essential or that excessive accumulation of ppGpp is lethal.     

A rifampicin resistance mutation in the rpoB gene confers ppGpp-independent antibiotic production in Streptomyces coelicolor A3(2)

In Streptomyces coelicolor A3(2), deletion of relA or a specific mutation in rplK ( relC) results in an inability to synthesize ppGpp (guanosine 5'-diphosphate 3'-diphosphate) and impairs production of actinorhodin. We have found that certain rifampicin-resistant ( rif) mutants isolated from either relA or relC strains regain the ability to produce actinorhodin at the same level as the wild-type strain, although their capacity to synthesize ppGpp is unchanged. These rif mutants were found to have a missense mutation in the rpoB gene that encodes the RNA polymerase beta-subunit. This rpoB mutation was shown to be responsible for the observed changes in phenotype, as demonstrated by gene replacement experiments. Gene expression analysis revealed that the restoration of actinorhodin production in both relA and relC strains is accompanied by increased expression of the pathway-specific regulator gene actII-ORF4, which is normally decreased in the rel mutants. In addition to the restoration of antibiotic production, the rif mutants also exhibited a lower rate of RNA synthesis compared to the parental strain when grown in a rich medium, suggesting that these mutant RNA polymerases behave like "stringent" RNA polymerases. These results indicate that rif mutations can alter gene expression patterns independently of ppGpp. We propose that RNA polymerases carrying particular rif mutations in the beta-subunit can functionally mimic the modification induced by binding of ppGpp.     

Escherichia coli mutant containing a large deletion from relA to argA.

A mutant of Escherichia coli has been isolated that contains a large deletion (about 3 X 10(7) daltons of deoxyribonucleic acid) encompassing argA, fuc, and relA. This mutant strain (AA-787) is also cold sensitive for growth at 18 degrees C. Strain AA-787 was obtained fortuitously as a cold-sensitive pseudorevertant of a strain having a heat-sensitive peptidyl-transfer ribonucleic acid hydrolase. Genetic analysis, using transduction and interrupted mating, showed the cold sensitivity mutation to be located adjacent to relA. Further analysis demonstrated loss of relA, fuc, and argA gene functions but retention of eno and recB, closely linked genes adjacent to relA and argA, respectively. Unusually high cotransduction of flanking markers (cysC and thyA) indicated loss of approximately 1 min of the E. coli genetic map in strain AA-787. Guanosine 3'-diphosphate 5'-diphosphate (ppGpp) was synthetized in mutant strain AA-787 at basal levels, and ppGpp synthesis was stimulated by carbon-source downshift. No ppGpp synthesis could be obtained using ribosomes isolated from strain AA-787. These findings, taken together, show that deletion of relA in E. coli does not completely abolish ppGpp synthesis and suggests that another enzyme system must also be responsible for ppGpp synthesis.     

Suppressive effect of l-phenylalanine on lignin peroxidase in the white-rot fungus Phanerochaete chrysosporium

Abstract Guanosine-5'-diphosphate-3'-diphosphate (ppGpp), an effector for many metabolic pathways, is synthesized by the relA gene product after amino acid limitation. Studies of stringent controlled Escherichia coli CP78 (relA⁺) and relaxed controlled E. coli CP79 (relA⁻) were carried out to test whether these strains differ in the appearance of their cytoplasmic membranes after induction of stringent and relaxed response. Cytoplasmic membrane structures of the cells were investigated by freeze-fracture electron microscopy after cooling the cells. The obtained micrographs showed a net-like distribution of the particles in the cytoplasmic membranes of relaxed controlled cells whereas such a pattern was not detectable in the stringent controlled counterparts.     

The regulation of stable RNA synthesis in the blue-green alga Anacystis nidulans: effect of leucine deprivation and 5-methyltryptophan inhibition.

The expression of phenomena associated with the bacterial function controlling RNA synthesis was studied in leucine-deprived or 5-methyltryptophan-treated cultures of Anacystis nidulans. Both procedures retarded cell growth, RNA and protein accumulation, elicited the accumulation of high intracellular concentrations of guanosine 5'-diphosphate-3'-diphosphate(ppGpp)and guanosine5'-triphosphate-3'-diphosphate(pppGpp),and promoted a regime of non-coordinate synthesis of stable and messenger RNA. The rate of polymerization of nascent RNA chains did not appear to be retarded in the growth-limited cultures.     

Accumulation of ppGpp in a relA mutant of Escherichia coli during amino acid starvation

In Escherichia coli, amino acid starvation triggers the rapid synthesis of two guanosine polyphosphates, pppGpp and ppGpp (the 3'-pyrophosphates of GTP and GDP, respectively). Determination of the turnover rate of the ppGpp pool indicated that during serine deprivation, as opposed to other amino acid starvations, the rate of ppGpp degradation is dramatically decreased. This results in a slow but significant accumulation of this regulatory nucleotide in a relA mutant during serine starvation. Similar ppGpp accumulation can be seen during serine starvation in different serine auxotrophic mutants carrying different relA alleles. On the other hand, no ppGpp accumulation is induced in various relaxed strains by serine hydroxamate treatment.